Expression, purification and characterisation of recombinant Escherichia coli derived chicken interleukin-12.
Zoonotic viruses, such as H5N1 Avian Influenza, pose major threats to both animals and humans, and with this in mind there is a need for the development of new anti-viral strategies. The cytokine interleukin-12 (IL-12) is known to play a pivotal regulatory role in the anti-viral response due to its role in the induction of the key anti-viral cytokine IFN-gamma. Therefore, strategies which provide a means for the production of therapeutic quantities of IL-12 may be of major benefit. Here we describe the development of biologically active Escherichia coli (E. coli) derived chicken IL-12 (ChIL-12). The single chain ChIL-12 gene was cloned into the pET32b expression vector, transformed into the BL-21 E. coli strain and expression induced with IPTG. Over expressed protein was solubilised with zwittergent detergent and isolated utilising Nickel ion affinity chromatography. Biological activity was determined as ChIL-12 stimulated proliferation of pre-treated T-cells in vitro. This study is the first example of a biologically active E. coli derived IL-12 from a non-mammalian vertebrate subsequently providing a means for testing the anti-viral therapeutic potential of ChIL-12 in an in vivo model.
Thomas
Jesse D
JD
CSIRO - Australian Animal Health Laboratories, Livestock Industries, Private Bag 24, Geelong, Victoria 3220, Australia.
Morris
Kirsten R
KR
Godfrey
Dale I
DI
Lowenthal
John W
JW
Bean
Andrew G D
AG
D000818:Animals; D002645:Chickens / Q000235:genetics* / Q000276:immunology; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004926:Escherichia coli; D005822:Genetic Vectors / Q000235:genetics*; D018664:Interleukin-12 / Q000235:genetics* / Q000378:metabolism*; D007544:Isopropyl Thiogalactoside
D018664:Interleukin-12; D007544:Isopropyl Thiogalactoside